Method of promoting osteogenesis

ABSTRACT

A METHOD OF PROMOTING OSTEOGENESIS IN ANIMAL HOSTS WHICH COMPRISES APPLYING TO THE AREA OF A BONE DEFECT A QUARTERNARY AMMONIUM COMPOUND OF THE FORMULA   (R-)2-N(+)(-(CH2)N-)-((CH2)N)-N(+)(-R)2 (X(-))2   WHEREIN R REPRESENTS LOWER ALKYL, X REPRESENTS CHLORINE OR BROMINE, AND N IS AN INTEGER FROM SIX TO TWELVE; OR OF THE FORMULA;   R1-N(+)(-R2)(-R3)-R4 X(-)   WHEREIN R4 REPRESENTS AN ALKYL GROUP CONTAINING TWELVE TO TWENTY CARBON ATOMS; R1, R2 AND R3 EACH REPRESENT LOWER ALKYL, AND R1, R2 AND R3 TAKEN TOGETHER WITH THE NITROGEN ATOM REPRESENT PYRIDIUM; AND X REPRESENTS CHLORINE OR BROMINE.

United States Patent 3,577,540 METHOD OF PROMOTING OSTEOGENESIS Dirck V.Myers, Atlanta, Ga., assignor to E. R. Squibb & Sons, Inc., New York,NY. No Drawing. Filed Nov. 12, 1968, Ser. No. 775,162 Int. Cl. A61k27/00 US. Cl. 424-244 8 Claims ABSTRACT OF THE DISCLOSURE A method ofpromoting osteogenesis in animal hosts which comprises applying to thearea of a bone defect a quaternary ammonium compound of the formulawherein R represents lower alkyl, X represents chlorine or bromine, andn is an integer from six to twelve; or of the formula 31 R 1 IR X-wherein R represents an alkyl group containing twelve to twenty carbonatoms; R R and R each represent lower alkyl, and R R and R takentogether with the nitrogen atom represent pyridinium; and X representschlorine or bromine.

the formula R (CH2)n R I wherein R represents lower alkyl, X representschlorine or bromine, and n is an integer from six to twelve; or

of the formula R Ir wherein R represents an alkyl group containingtwelve to twenty carbon atoms; R R and R each represent lower alkyl, andR R and R taken together with the nitrogen atom represent pyridinium;and X represents chlorine or bromine.

The term lower alkyl as employed herein includes both straight andbranched chain radicals of less than eight carbon atoms. Preferredcompounds for use in accordance with this invention comprise compoundsof Formula I in which all four R groups represent methyl, and compoundsin accordance With Formula 11 wherein R represents a straight chainalkyl group.

Compounds of Formula I may be prepared by a twostep process. In thefirst step, a polymethylene dichloride or dibromide containing six totwelve carbon atoms is reacted with a di(lower alkyl)amine in thepresence of a solvent such as absolute alcohol to produce acorresponding polymethylene-bis-di(lower alkyl) amine. This is thenreacted in the second step of the process with an equimolar amount ofthe polymethylene dihalide to produce the quaternary ammonium compoundsof this invention.

For use, the quaternary ammonium compounds of this invention arepreferably suspended in a sterile physiological saline, e.g., about 2.0to about 60 mg./ml. This is administered by injection into the areaimmediately adjacent or surrounding the site in the animal host (e.g.,rats, dogs, cats, cattle, horses), at which it is desired to promote orstimulate bone growth, for example, at the site of bone implants,fractures, or in various metabolic bone defects (osteoporosis), orapplication to the periodontal tissue. In general, about 1 to 30 mg.,preferably about 2 to 5 mg. of octeogenic agent per kg. of body weightis injected. This may be repeated one or more times as required.Alternatively, in the case of bone implants, a solution (for instance, 1mg./ml.) can be made in sterile water and implant material can besaturated therewith as by immersion before being used in the patient.

The following examples illustrate the invention, all temperatures beingin degrees centigrade.

EXAMPLE 1 (a) Bis-1,10-dimethylaminodecane Into a pint-sized pressurebottle were placed 30.0 g. of 1,10-decarnethylene dibromide, 18.0 g.dimethylamine, and 150 ml. absolute alcohol. The bottle was sealed andallowed to stand for five days. It was heated intermittently to 50. Thecontents were poured into a 2.1. Erlenmeyer flask and diluted with 750ml. anhydrous ether. A crystalline material was obtained, dimethylaminehydrobromide. It was removed by filtration and the filtrate wasconcentrated. The residue was distilled to give 17.5 g. of product, B.P.143-146 (8 mm.)

(b) 1,1,12,12-tetramethyl-1,12-diazacyclodocosane dibromide Into apressure bottle were placed 16.9 g. of the product of Example 1(a), 24.3g. decamethylene dibromide and 150 ml. acetone. The mixed materials wereallowed to stand twelve days. The product was filtered andrecrystallized from 200 ml. absolute alcohol and 1200 ml. acetone togive 27.9 g. of product, M.P. 193-194". The product is quitehygroscopic.

EXAMPLE 2 1,1,8,8-tetramethyl-1,8-diazacyclotetradecane dibromideFollowing the procedure of Example 1(b) but substituting 17 g. ofbis-1,6-dimethylaminohexane for the bis-1,10- dimethylaminodecane and24.5 g. of hexamethylene dibromide for the decamethylene dibromide,there is obtained 27 g. of product, M.P. 2525-2535".

EXAMPLE 3 Sr assay of osteogenic agents The osteogenic agents of thisinvention are assayed by suspending a 60 mg. sample in 1 ml. ofphysiological saline and the pH is adjusted within the range of 6 to 7.

The ostegoenic agent thus suspended is injected (0.1 ml., 6 mg. dose)along the periosteal surface of the radiusulna complex of male ratsweighing 180200 g. The contrallateral radius-ulna complex serves as thecontrol. The rats are maintained for one week at which time 2 microcuries of radioactive Sr preferably as Sr nitrate, is injectedintrathoracically. On the following day the animals are sacrificed andthe Sr content of the treated and control radius-ulna complex ismeasured. The assay depends upon the enhanced ability of newly formedbone (matured osteoid) to bind Sr in addition to that bound by thepre-existing bone. The extent of bone growth induced within the week byosteogenic material is determined by calculating the difierence inuptake of Sr as calculated by the following expression:

A value of 100 corresponds to no excess incorporation of Sr in thetreated complex as compared with the control. Values above 100 give ameasure of the degree of osteogenic potency of the materials (per mg. ofdose) injected.

New bone production is always accompanied by a soft tissue reaction.This reaction varies with the material used to induce osteogenesis andmay be separated histologically into an acceptable and an unacceptablecategory.

An acceptable tissue reaction is described as follows: Eight daysfollowing the injection of the osteogenic factor preparation thereexists shrinkage and lysis of some muscle bundles. There is an influx oflarge numbers of fibroblasts associated with considerable new bloodvessel formation. These fibroblasts (precursor cells) arenon-differentiated; however, some do differentiate into osteoblasts inthe area of new bone formation. A few giant cells are usually present inthese areas and no indication of abscess formation exists. The new boneformation is quite extensive and occurs radially around the radius andulna. The new bone is not compact but appears to be cancellous in naturewith the formation of many discrete bone marrow cavities. This is thetype of histological response which accompanies normal bone formation(e.g., repair of fractures).

Abnormal undesirable tissue reactions are described as follows: Eightdays following the injection of certain sub stances there is shrinkage,lysis and necrosis of many muscle bundles. Abscess formation existsaccompanied by massive tissue destruction. Fibroplasia does occur;however, it is not extensive. New bone formation occurs radially aroundthe radius and the ulna. Resorption of the original shaft of the radiusand ulna exists, often accompanied by rechanneling of the marrow cavity.

The Sr ratio and histological response observed in rat legs injectedwith compounds of this invention are tabulated below. In all cases, thehistological response is acceptable, the new bone formation beingcancellous in nature with the formation of many discrete bone marrowcavities. No resorption of the original bone shaft has taken place.

wherein R represents lower alkyl, X is selected from the groupconsisting of chlorine and bromine, and n is an integer from six totwelve, and compounds of the formula a- R wherein R represents an alkylgroup containing twelve to twenty carbon atoms; R R and R are each loweralkyl; R R and R taken together with a nitrogen atom is pyridinium; andX is selected from the group consisting of chlorine and bromine.

2. A method in accordance with claim 1 wherein the compound administeredhas the formula R\ )CHQH R H N N R/ (C2)n R wherein R, X and n are asset forth in claim 1.

3. A method in accordance with claim 1 wherein the compound administeredhas the formula R1 R2 Is -N R3 wherein R R R and R and X are as setforth in claim 1.

4. A method in accordance with claim 1 wherein the quaternary ammoniumcompound is administered in an amount of from about 1 to about 30 mg.per kg. of body weight.

5. A process in accordance with claim 2 wherein the compoundadministered is 1,1,12,12 tetramethy1-l,12- diazacyclodocosanedibromide.

6. A method in accordance with claim 2 wherein the compound administeredis 1,1,8,8-tetramethyl-l,8-diazacyclotetradecane dibromide.

7. A method in accordance with claim 3 wherein the compound administeredis hexadecyltrimethylammonium bromide.

8. A method in accordance with claim 3 wherein the compound administeredis l-hexadecylpyridinium chloride.

Dose New bone Undifieren- Giant Abcess Compound (mg/kg.) (Sr ratio)Fibroplasia tiated cells cells Necrosis formation 0E3 )CEzho/CHQ N N -2Br- 2.0 267 Extensive." Yes Yes None None. CH3 (OHz)1o CH3 CH3 (OHM CH3-2 Br- 2.3 Do. CH3 (CHz)o CH3 CH:4(CHz) 5-N -Cl- 3.7 D0.

CHs( 2)1s s)a-B 2- D0.

Same as above. 2. 5 D0. D0 1.7 D0. D0 0.8 Do. Do 0.4 Do.

References Cited UNITED STATES PATENTS 2,899,357 8/1959 Cavallito et al.424-329 2,977,280 3/1961 Forsyth et al 424329 3,034,965 5/1962 Drake eta1. 424329 3,227,614 1/1966 Scheuer 424-329 STANLEY J. FRIEDMAN, PrimaryExaminer D. M. STEPHENS, Assistant Examiner U.S. Cl. X.R. 424-329

